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En el artículo se describen y analizan los resultados de un estudio realizado para investigar la relación entre la práctica físico-deportiva y la calidad de vida percibida (CVP). La muestra del estudio estuvo integrada por un colectivo de ciudadanos (968), pacientes de Atención Primaria, entrevistados mientras se encontraban en las salas de espera, de cuatro Centros de Atención Primaria (CAP) de la ciudad de Terrassa. Los objetivos del estudio fueron: 1) Evaluar la relación entre la percepción de calidad de vida y la actividad físico-deportiva; 2) Verificar si, en tal percepción, hay diferencias significativas entre hombres y mujeres. Las conclusiones fueron las siguientes: Existe una percepción mejor de la calidad de vida entre los usuarios que realizan actividad físico-deportiva que entre los pacientes sedentarios, sea la actividad moderada o intensa. Las mujeres que realizan actividad físico-deportiva mejoran su percepción de Calidad de Vida en mayor grado que los hombres. Las mujeres mejoran su CVP en el ámbito social y los hombres en el ámbito físico, por lo que la práctica deportiva es una fuente de la socialización de las mujeres. Abstract. The article describes and analyzes the results of a study carried out to investigate the relationship between physical-sports practice and perceived quality of life (QOL). The study sample was made up of a group of citizens (968), Primary Care patients, interviewed while they were in the waiting rooms of four Primary Care Centers (CAP) in the city of Terrassa. The objectives of the study were: 1) To evaluate the relationship between the perception of quality of life and physical-sports activity; 2) Verify if, in such perception, there are significant differences between men and women. The conclusions were as follows: There is a better perception of the quality of life among users who perform physical-sports activity than among sedentary patients, be it moderate or intense activity. Women who perform physical-sports activity improve their perception of Quality of Life to a greater degree than men. Women improve their CVP in the social sphere and men in the physical sphere, so sports practice is a source of women's socialization.

Public health is funded with government funds gathered from tax revenues, whether national, provincial or municipal. The health system therefore suffers during economic crisis periods, whether due to disinvestment, loss of purchasing power among health care personnel or the decrease in the number of professionals. This worsens the situation, as it is necessary to cover the needs of an increasingly elderly population and with a longer life expectancy at birth. The present study intends to show a model which explains the determination of the “Public Health Personnel Expenditure” in Spain for a determined period. A multiple linear regression model was applied to the period including the years 1980–2021. Macroeconomic and demographic variables were analyzed to explain the dependent variable. Variation in health personnel expenditure: “We included those variables which presented a high or very high correlation above r > 0.6. The variables which explain the behavior of Variation in health personnel expenditure”. It was a determining factor in the present study to consider that the variables with the greatest repercussions on health policy were mainly macroeconomic variables rather than demographic variables, with the only significant demographic variable that had a specific weight lower than macroeconomic variables being “Birth Rate”. In this sense, the contribution made to the scientific literature is to establish an explanatory model so that public policy managers and states in particular can consider it in their public spending policies, bearing in mind that health expenditures in a Beveridge-style health system, as Spain has, are paid with funds drawn from tax revenues.

This article analyzes the relationship between per capita expenditure and financial and macroeconomic variables in the framework of mental health, in regions where the prevailing system is public healthcare governed by the state and in regions where the prevailing system is that of public ownership. The period 2006–2017 was analyzed. A simple linear regression analysis was carried out to determine the relationship between the expenditure per inhabitant and a series of relevant variables such as asset turnover, cash flow, and expenditure as a percentage of gross domestic product (GDP), applying statistical tests to validate the study. In regions where public–private co-financing prevails in the health system, two crucial variables to measure per capita expenditure on mental health were GDP per capita and cash flow of mental health providers. In the regions where management is direct, the crucial variables were asset turnover of mental health providers and expenditure on mental health as a percentage of GDP per capita. These elements are key to determining how to develop public investment policies in hospital systems in the different regions of Europe and the world.

En el presente artículo se recogen las conclusiones de un estudio que analiza la relación entre la práctica físico-deportiva de los usuarios de centros deportivos públicos de la ciudad de Terrassa (provincia de Barcelona) y la calidad de vida percibida por los mismos, estudio realizado sobre una muestra de 674 usuarios practicantes de actividad física en ocho centros deportivos públicos abiertos en el momento de la recogida de información. Analizando la variable calidad de vida percibida, el equipo investigador ha logrado definir perfiles de usuarios de los centros deportivos públicos de Terrassa y medir cómo éstos perciben su calidad de vida, habiendo podido distinguir colectivos que no están siendo atendidos actualmente por tales centros deportivos o que lo están siendo de manera incorrecta o insuficiente. Las conclusiones del estudio permitirán al Ayuntamiento de Terrassa una mejor orientación de su política pública en materia de actividad físico-deportiva, en función de las características de sus usuarios/clientes y de su percepción sobre el impacto que tal actividad físico-deportiva practicada en las condiciones actuales tiene en su calidad de vida percibida, es decir, en función de la demanda actual y latente.Abstract. This study analyzes the relationship between the physical-sports practice of users of public sports centers in the city of Terrassa and their perceived quality of life, in a sample of 674 users who practice physical activity in eight public sports centers open at the time of the data collection. Analyzing the variable “perceived quality of life”, the research team managed to define user profiles of the public sports centers of Terrassa and measure how they perceive their quality of life, defining groups that are either not currently being attended or that are being improperly treated by the current centers. This will allow the City Council to orient its public policy in the area of physical and sports activity, given the characteristics of its users / customers and the perceived quality of life, that is, based on current and latent demand.

Purpose: The present study identifies the professional skills profile that a Public Affairs (PA) practitioner of a major company or an organization representing the interests of various economic sectors should have in Spain. Design/methodology: The study uses a mixed qualitative and quantitative methodology. The initial identification of the competences, as well as the general functions of the profile to be evaluated, has been carried out using the in-depth interview technique with the directors of APRI (Association of Professionals of Institutional Relations of Spain), who defined these competencies from their expert perspective. Subsequently, through a survey of seventy-two managers, sufficient data were obtained to statistically process the information and obtain significant results in the identification of such competences. This information has been processed, firstly, through factorial analysis, which has made it possible to synthesize these competences. Once the analysis of factors was carried out, a cluster analysis is added for the classification of the respondents or managers. Findings: The perception of the interviewed executives has been determined regarding the functions that an institutional relations practitioner must fulfill. It should be noted that this is a profession for which there is no clarity about the role and the legitimacy of its members, particularly in Spain. In fact, it is associated with a function that is seen as exerting undue pressure on political power. A field study was conducted to determine the opinion of these managers on the competences that a professional in institutional relations should have. Originality/value: This study provides a description of the job position of a person responsible for institutional relations in Spain. Moreover, it adds a typology of managers, according to the competences defined for the person responsible for institutional relations.

The rapid evolution of antibiotic resistance in Clostridium difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matter of concern for public health. Antibiotic resistance plays an important role in driving C. difficile epidemiology. Emergence of new types is often associated with the emergence of new resistances and most of epidemic C. difficile clinical isolates is currently resistant to multiple antibiotics. In particular, it is to worth to note the recent identification of strains with reduced susceptibility to the first-line antibiotics for CDI treatment and/or for relapsing infections. Antibiotic resistance in C. difficile has a multifactorial nature. Acquisition of genetic elements and alterations of the antibiotic target sites, as well as other factors, such as variations in the metabolic pathways and biofilm production, contribute to the survival of this pathogen in the presence of antibiotics. Different transfer mechanisms facilitate the spread of mobile elements among C. difficile strains and between C. difficile and other species. Furthermore, recent data indicate that both genetic elements and alterations in the antibiotic targets can be maintained in C. difficile regardless of the burden imposed on fitness, and therefore resistances may persist in C. difficile population in absence of antibiotic selective pressure.

The rapid evolution of antibiotic resistance in Clostridium difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matter of concern for public health. Antibiotic resistance plays an important role in driving C. difficile epidemiology. Emergence of new types is often associated with the emergence of new resistances and most of epidemic C. difficile clinical isolates is currently resistant to multiple antibiotics. In particular, it is to worth to note the recent identification of strains with reduced susceptibility to the first-line antibiotics for CDI treatment and/or for relapsing infections. Antibiotic resistance in C. difficile has a multifactorial nature. Acquisition of genetic elements and alterations of the antibiotic target sites, as well as other factors, such as variations in the metabolic pathways and biofilm production, contribute to the survival of this pathogen in the presence of antibiotics. Different transfer mechanisms facilitate the spread of mobile elements among C. difficile strains and between C. difficile and other species. Furthermore, recent data indicate that both genetic elements and alterations in the antibiotic targets can be maintained in C. difficile regardless of the burden imposed on fitness, and therefore resistances may persist in C. difficile population in absence of antibiotic selective pressure.

Recent studies suggest animals, in particular farm and companion animals, as possible reservoir for Clostridium difficile human pathogenic strains. The aim of this study was to give a first characterization of C. difficile isolates from Italian swine and dogs. In total, 10 different PCR-ribotypes were identified among porcine strains and six among canine strains. The predominant type found among porcine strains was 078 (50%), whereas the most frequently detected among canine strains was the non-toxinogenic 010 (64%). Considering the CLSI breakpoints, 60% of porcine isolates was resistant to ERY, 35% to MXF, 15% to CLI, 5% to RIF, and none to MTZ or VAN. Among dogs, 51% of strains was resistant to CLI, 46% to ERY, 21% to MTZ and 5% to MXF or RIF, and none to VAN. Five porcine strains (10%) and 9 canine isolates (41%) were MDR. Interestingly, 8 MDR canine strains were highly resistant to MTZ, with MICs ≥32 mg/L. Considering the EUCAST cut-off for MTZ (MIC >2 mg/L), 13 canine isolates and one porcine strain were found with reduced susceptibility to MTZ (MICs ranging from 3 to ≥256 mg/L). Swine and canine strains showing resistance or reduced susceptibility to MTZ belonged to PCR-ribotype 010 and 078. These PCR-ribotypes have been associated to reduced susceptibility to MTZ also in human, suggesting a potential risk for the emergence of C. difficile strains resistant to the current first-line antibiotic for CDI treatment. The agar incorporation method (AIM) was confirmed as the best method to detect C. difficile strains with this phenotype also after strains manipulations. The results obtained add further evidences about the possible role of animals as source of MDR C. difficile strains and reservoir of antibiotic resistance determinants.

Point mutations conferring resistance to fluoroquinolones were introduced in the gyr genes of the reference strain Clostridium difficile 630. Only mutants with the substitution Thr-82→Ile in GyrA, which characterizes the hypervirulent epidemic clone III/027/NAP1, were resistant to all fluoroquinolones tested. The absence of a fitness cost in vitro for the most frequent mutations detected in resistant clinical isolates suggests that resistance will be maintained even in the absence of antibiotic pressure.

In Clostridium difficile, resistance to the macrolide-lincosamide-streptogramin B group of antibiotics generally relies on erm(B) genes. In this study, we investigated elements with a genetic organization different from Tn5398, the mobilizable non-conjugative element identified in C. difficile strain 630. Our results suggested that the elements most frequently found in strains isolated during the European surveillance study in 2005 were related to Tn6194, the conjugative transposon recently detected in different C. difficile types, including PCR-ribotype 027. We characterized a Tn6194-like and a novel element rarely found in clinical isolates. A burden on the in vitro fitness of C. difficile was observed after the acquisition of these elements as well as of Tn5398.

Como hacer un Plan de Empresa Euncet pretende ser una herramienta de soporte donde poder encontrar toda la informacion imprescindible para elaborar un plan de negocio. El libro se presenta estructurado en tres bloques de contenido. El primero esta enfocado al planteamiento de la empresa; en el se trabaja, por un lado, la propuesta de negocio desde la idea inicial hasta el diseno del modelo de empresa y, por otro lado, el analisis estrategico, contemplando los diferentes entornos a considerar y proponiendo una planificacion estrategica de la empresa. El segundo bloque lo integran los diferentes planes operativos y estrategicos de la empresa. Asi pues, encontramos toda la informacion relativa a la elaboracion del Plan de Marketing, de Operaciones, de Recursos Humanos, Juridico-Fiscal, Economico-Financiero, de Contingencias y de Continuidad del Negocio. Finalmente, hay un tercer bloque compuesto por material extraordinario, a modo de anexos, indices y una amplia bibliografia sobre las diferentes cuestiones abordadas en el libro, asi como un glosario de terminos relacionados con el ambito empresarial.

The aim of this study was to investigate the S-layer proteins (SLPs) of the hypervirulent Clostridium difficile PCR ribotype 027 and compare them with those of PCR ribotype 001 and other PCR ribotypes involved in C. difficile infection and outbreaks, by molecular analysis and immunological assays. It has been demonstrated previously that PCR ribotype 027 SlpA is conserved in C. difficile strains belonging to this PCR ribotype and that it is a new variant, showing 88 % identity with SlpA of PCR ribotype 001. As the low-molecular-weight (LMW) SLPs of C. difficile are immunodominant antigens, attention was focused on this region of the genome. Sequencing of strains of different PCR ribotypes (001, 012, 014, 017, 027 and 078) showed that SlpA was conserved among strains belonging to the same PCR ribotype. Comparison of the LMW SLP region among these strains identified ten regions with sequence identity between PCR ribotypes 027 and 001, and low conservation with the other PCR ribotypes. In particular, two of these regions corresponded to areas predicted to be surface exposed. Three specific peptides, including those of the two surface-exposed regions, were recognized by human sera against PCR ribotypes 027 and 001 and by a rabbit polyclonal serum against the SLPs of PCR ribotype 027. In contrast, these peptides were not recognized by a polyclonal serum against the SLPs of PCR ribotype 012 used as a control. These results confirm the antigenic role of the LMW SLP and suggest that it may have a role in evasion of the host immune response.

Recent evidence strongly suggests an association between the use of fluoroquinolones and Clostridium difficile infection (CDI). Resistance to fluoroquinolones has been described not only in the hypervirulent strain 027, but also in other important PCR ribotypes circulating in hospital settings. In a European prospective study conducted in 2005, strains resistant to moxifloxacin represented 37.5% of C. difficile clinical isolates. In this study, we investigated a sample of 147 toxigenic C. difficile isolates, collected in Italy from 1985 to 2008, for the presence of mutations in gyr genes that conferred resistance to fluoroquinolones based on a LightCycler assay. Results were confirmed by the determination of MICs for moxifloxacin. Strains resistant to moxifloxacin were also investigated for resistance to three other fluoroquinolones and for a possible association between fluoroquinolone and macrolide-lincosamide-streptogramin B resistance. C. difficile isolates were typed by PCR ribotyping. In total, 50 clinical isolates showed substitutions in gyr genes and were resistant to fluoroquinolones. Ninety-six percent of the C. difficile resistant isolates showed the substitution Thr82-to-Ile in GyrA, as already observed in the majority of resistant strains worldwide. A significant increase of resistance ( P < 0.001) was observed in the period 2002 to 2008 (56% resistant) compared to the period 1985 to 2001 (10% resistant). Coresistance with erythromycin and/or clindamycin was found in 96% (48/50) of the isolates analyzed and, interestingly, 84% of resistant strains were erm (B) negative. The majority of the fluoroquinolone-resistant isolates belonged to PCR ribotype 126 or 018. PCR ribotype 126 was the most frequently found from 2002 to 2005, whereas PCR ribotype 018 was predominant in 2007 and 2008 and still represents the majority of strains typed in our laboratory. Overall, the results demonstrate an increasing number of C. difficile strains resistant to fluoroquinolones in Italy and changes in the prevalence and type of C. difficile isolates resistant to fluoroquinolones circulating over time.

Recent outbreaks of Clostridium difficile infections (CDI), with increased severity, high relapse rates, and significant mortality, have been related to the emergence of the hypervirulent C. difficile clone toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 (5, 23, 25-29, 31). Several studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of CDI caused by C. difficile III/027/NAP1 and that the acquisition of resistance to the newer fluoroquinolones could have promoted its wide dissemination (6, 17, 30, 32-34). Fluoroquinolones are a family of broad-spectrum antibiotics extensively used in the treatment of a great variety of human infections. The in vitro activity of the older fluoroquinolones, such as ciprofloxacin, has been reported to be moderate or poor against anaerobes, including C. difficile (3, 8), whereas the third and the fourth generations of fluoroquinolones are characterized by improved activity against gram-positive cocci and anaerobic bacteria (19, 36). Fluoroquinolones act by inhibiting the action of DNA gyrase and topoisomerase IV, which are related but distinct enzymes involved in DNA synthesis (18). The mechanisms of resistance to fluoroquinolones in bacteria are basically two: (i) alterations in the targets of fluoroquinolones and (ii) decreased accumulation inside the bacteria due to impermeability of the membrane and/or an overexpression of efflux pump systems (19, 20, 36). The first mechanism of resistance is widespread in many bacteria, and it is due to amino acid substitutions in the quinolone-resistance determining region (QRDR) of the target enzymes (35). This is the principal mechanism of resistance also in C. difficile, and since, as already observed in other species, this bacterium does not have genes for topoisomerase IV, resistance is determined by alterations in the QRDR of either DNA gyrase subunit GyrA or GyrB (10, 38). Different amino acid substitutions have been identified in GyrA and GyrB in fluoroquinolone-resistant C. difficile strains. The most frequent is the amino acid change Thr82 to Ile in GyrA, which also characterizes the epidemic clone III/027/NAP1 (11, 38). Two other GyrA substitutions, Asp71 to Val and Ala118 to Thr, have been more rarely observed (1, 2, 10, 12, 38). Four different amino acid substitutions have been identified in GyrB: Arg447 to Lys, Arg447 to Leu, Asp426 to Asn, and Asp426 to Val (10, 11, 38). In particular, Asp426 to Val has been described in toxin A-negative/toxin B-positive C. difficile epidemic strains of recent isolation (11). In this study, we evaluated the potential of moxifloxacin (MX) and levofloxacin (LE), recently associated with outbreaks caused by C. difficile III/027/NAP1 (25, 31, 33), for the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations in five different susceptible C. difficile strains. The sequence changes occurring in the QRDR of the derived fluoroquinolone-resistant mutants were analyzed and correlated with the in vitro resistance to MX, LE, and gatifloxacin (GA), another fluoroquinolone recently involved in C. difficile outbreaks (17, 33).

We report a noteworthy case of Eubacterium plautii infection after kidney transplantation. Our 33-yr-old transplant recipient received standard care; his post-transplant course was uneventful. However, on day 44 he underwent an emergency laparotomy for perforation of the ileum. He was initially treated with ceftazidime, fluconazole and metronidazole, but his fever persisted, so he was switched to meropenem and vancocin. We could not find any cause for his infection. On day 70, his temperature normalized. On day 75, he developed severe leukopenia (280 cell/mL). His cytomegalovirus-DNA test result was negative, so all immunosuppressants, except for prednisone, were stopped; instead, antibiotic prophylaxis was started, using caspofungin, trimethoprim-sulfamethoxazole and ciprofloxacin. On day 83, he underwent percutaneous drainage of massive left pleural effusion. We repeatedly cultured the pleural liquid, but it was not till three wk later that we were finally able to identify the causative organism. We hypothesize that the microorganism - which normally resides on the surface of the intestinal lumen - entered the bloodstream via bacterial translocation, eventually colonizing the pleurae. This translocation was favored by our patient poor clinical condition, his immunosuppressive treatment and his heavy antibiotherapy. Our experience highlights the need for wiser use of antibiotics in transplant recipients.

A 2-month prospective study of Clostridium difficile infections was conducted in 38 hospitals from 14 different European countries in order to obtain an overview of the phenotypic and genotypic features of clinical isolates of C. difficile during 2005. Of 411 isolates from diarrhoeagenic patients with suspected C. difficile-associated diarrhoea (CDAD), 354 were toxigenic, of which 86 (24.3%) were toxin-variant strains. Major toxinotypes included toxinotypes 0 (n = 268), V (n = 28), VIII (n = 22) and III (n = 25). MICs of metronidazole, vancomycin, erythromycin, clindamycin, moxifloxacin and tetracycline were determined using the Etest method. All the toxigenic strains were fully-susceptible to metronidazole and vancomycin. Resistance to erythromycin, clindamycin, tetracycline and moxifloxacin was found in 44.4%, 46.1%, 9.2% and 37.5% of the isolates, respectively. Sixty-six different PCR ribotypes were characterised, with the 027 epidemic strain accounting for 6.2% of isolates. This strain was positive for binary toxin genes, had an 18-bp deletion in the tcdC gene, and was resistant to both erythromycin and moxifloxacin. The mean incidence of CDAD was 2.45 cases/10 000 patient-days, but this figure varied widely among the participating hospitals. Patients infected with the 027 strain were more likely to have a severe disease (OR 3.29, 95% CI 1.19–9.16, p 0.008) and to have been specifically treated with metronidazole or vancomycin (OR 7.46, 95% CI 1.02–154, p 0.02). Ongoing epidemiological surveillance of cases of CDAD, with periodic characterisation of the strains involved, is required to detect clustering of cases in time and space and to monitor the emergence of specific highly virulent clones.

This study describes the first two reported invasive nontypeable Haemophilus influenzae (NTHI) isolates (strains 183 and 184) with heterogeneous resistance to imipenem. For both isolates, Etest showed imipenem MICs of ≥32 μg/ml. When the two strains were examined by the quantitative method of population analysis, both strain populations were heterogeneously resistant to imipenem and contained subpopulations growing in the presence of up to 32 μg of imipenem/ml at frequencies of 1.7 × 10 −5 and 1.5 × 10 −7 , respectively. By pulsed-field gel electrophoresis analysis, the two isolates appeared to be genetically closely related. The sequencing of the ftsI gene encoding penicillin-binding protein 3 (PBP 3) and comparison with the sequence of the imipenem-susceptible H. influenzae strain Rd identified a pattern of six amino acid substitutions shared between strains 183 and 184; an additional change was unique to strain 183. No relationship between mutations in the dacB gene encoding PBP 4 and imipenem resistance was found. The replacement of the ftsI gene in the imipenem-susceptible strain Rd (for which the MIC of imipenem is 0.38 to 1 μg/ml) with ftsI from strain 183 resulted in a transformant for which the MIC of imipenem ranged from 4 to 8 μg/ml as determined by Etest. The Rd/183 transformant population showed heterogeneous resistance to imipenem; it contained subpopulations growing in the presence of up to 32 μg of imipenem/ml at a frequency of 3.3 ×10 −8 . The presence of additional resistance mechanisms, such as the overexpression of the AcrAB efflux pump, was investigated and does not seem to be involved. These data indicate that the heterogeneous imipenem resistance phenotype of our NTHI clone depends largely on the PBP 3 amino acid substitutions. We speculated that bacterial regulatory networks may play a role in the control of the heterogeneous expression of the resistance phenotype.

Elements carrying more than one antibiotic resistance gene have never been found in Clostridium difficile, one of the major causes of nosocomial diarrheic diseases. In this study, C. difficile isolates were investigated for a possible genetic linkage between tet(M) and erm(B), the most frequent genes found in strains resistant to tetracycline and erythromycin. In the majority of C. difficile strains, tet(M) is carried by Tn5397. However, tet(M) genes carried by Tn916-like elements have been found in recent clinical isolates. As far as erythromycin resistance is concerned, the only completely characterized transposon harboring an erm(B) gene in C. difficile is Tn5398, even if ErmB determinants probably carried by other elements have been identified. Among the 100 C. difficile isolates screened in this study, 27 were positive for tet(M) and erm(B). Twenty five of these strains were positive for tndX, used as marker for Tn5397, whereas two were positive for int, used as marker for Tn916-like elements. The latter isolates showed two tet(M) genes: one was carried by a Tn916-like element, able to transfer to a recipient C. difficile strain, whereas the second was genetically linked to an erm(B) in a composite element probably unable to conjugate. Molecular analysis of C. difficile cd1911 tet(M)-erm(B) DNA sequence demonstrated that this region has arisen by recombination of DNA fragments from different plasmids and transposons. This is the first demonstration that C. difficile is able to accumulate and maintain antibiotic resistance genes, as observed in other pathogens.

Meningococcal invasive disease is a life-threatening infection that affects mostly children and adolescents. The present study was performed during 2003–2005 to compare the phenotypic characteristics of meningococcal isolates from these two main groups at risk with those of isolates from other age groups to assess whether strategies for treatment and prevention implemented elsewhere can also be applied in Italy. The results showed that serogroup C meningococci were predominant, and that a dramatic increase in the circulation of strains with decreased susceptibility to penicillin was associated mainly with a prevalent phenotype C:2b:P1.5,2, which belongs to the hyper-virulent ST8/A4 cluster.

A laboratory diagnosis survey of Clostridium difficile infection (CDI) was performed in Italy in 2012-2013. Questionnaires from 278 healthcare settings from 15 regions of Italy were collected and analysed. Eighty seven percent of the laboratories declared to routinely perform CDI diagnosis, 99% of them only after the clinician's request. Among the 216 laboratories providing information on the size of the hospitals in which they were located, 65 had more than 500 beds (large hospitals), while 151 had less than 500 beds (small hospitals). The average percentage of positive tests for C. difficile toxins was 12.2%. Almost half of the laboratories (42%) used immunoenzymatic assay (EIA) for Tox A/B as a stand-alone method, while only 34% used an algorithm for CDI as indicated by the European guidelines. A low percentage of laboratories performed molecular assays or C. difficile culture, 25% and 29%, respectively. Most laboratories (161/278) declared to type C. difficile strains, the majority in collaboration with a reference laboratory. Among the 103 C. difficile clinical isolates collected during the study, 31 different PCR-ribotypes were identified. PCR-ribotype 356/607 (27%) was predominant, followed by 018 (12%). These two PCR-ribotypes show 87.5% of similarity in ribotyping profile. PCR-ribotypes 027 and 078 represented 8% and 4% of the strains, respectively. Four PCR-ribotypes (027, 033, 078 and 126) were positive for the binary toxin CDT. In particular, PCR-ribotype 033 produces only CDT, and it has recently been associated with symptomatic cases. The majority of strains were multidrug resistant. In particular, all strains PCR-ribotypes 356/607 and 018 were resistant to moxifloxacin, rifampicin, erythromycin and clindamycin. The results obtained highlight the need to raise awareness to the microbiological diagnosis of CDI among clinicians and to implement and harmonize diagnostic methods for CDI in Italian laboratories in the perspective of a future national surveillance.

International audience; In Clostridium difficile, erm(B) genes are located on mobile elements like Tn5398 and Tn6215. In previous studies, some of these elements were transferred by conjugation-like mechanisms, mobilized in trans by helper conjugative systems. In this study, we analyzed the genomes of several recipient strains that acquired either Tn5398 or Tn6215-like elements. We demonstrated that the integration of the transposons in the genome of the recipient cell was always due to homologous recombination events, involving exchange of large chromosomal segments. We did not observed transposon transfer to a C. difficile strain in presence of DNAse, suggesting that a possible transformation-like mechanism occurred in this recipient.

Clostridium difficile , an etiological agent of most cases of antibiotic-associated diarrhea, exerts its pathological action mainly by the activity of toxin A and toxin B. Less known is the role that S-layer proteins (SLPs), predominant surface components of the bacterium, may play in pathogenesis. Here, we evaluate the ability of SLPs to modulate the function of human monocytes and dendritic cells (DC) and to induce inflammatory and regulatory cytokines, influencing the natural and adaptive immune response. To this aim, SLPs were extracted from the clinical isolate C253 and characterized for their effects on immune cells. SLPs induced the release of elevated amounts of interleukin (IL)-1β and IL-6 pro-inflammatory cytokines by resting monocytes, induced maturation of human monocyte-derived DC (MDDC), and enhanced proliferation of allogeneic T cells. C253-SLP-treated MDDC also secreted large amounts of IL-10 and IL-12p70 and induced a mixed Th1/Th2 orientation of immune response in naive CD4 T cells. In conclusion, C. difficile SLPs may contribute to the pathogenicity of the bacterium by perturbing the fine balance of inflammatory and regulatory cytokines. These data are of interest also in the light of the possible use of SLPs in a multicomponent vaccine against C. difficile infections for high-risk patients.

In this study, we set up a real-time reverse transcriptase PCR assay to measure the relative amounts of brkA transcripts in 50 Bordetella isolates. The results suggested that brkA expression is strain dependent and its level may play a role in determining the serum resistance or susceptibility phenotype. Pertussis immunocompetent sera were unable to kill Bordetella parapertussis via complement deposition.

This study demonstrates for the first time the in vitro transfer of the erythromycin resistance gene erm (B) between two obligate anaerobes, the human spore-forming pathogen Clostridium difficile and the rumen commensal Butyrivibrio fibrisolvens , suggesting that this event might occur also in the natural environment.

Most invasive Haemophilus influenzae type b strains possess a duplication of the capsulation locus. Further amplification resulting in as many as 5 copies has been described. To verify whether amplification is involved in vaccine failure, the number of copies of the locus was determined by Southern blotting in 90 strains from children with true vaccine failure (TVF) between 1993 and 1999 and in 139 strains from unvaccinated children (50 collected between 1993 and 1999 and 89 collected between 1991 and 1992, before routine immunization was introduced). A significantly greater proportion of strains from TVFs contained multiple copies, compared with strains from control children (24% vs. 10%; P = .0379), which suggests that amplification of the capb locus may be a contributory factor in vaccine failure. The presence of multiple-copy strains was associated with disease other than meningitis.

Bordetella pertussis, the causative agent of whooping cough, is internalized by several cell types, including epithelial cells, monocytes, and neutrophils. Although its ability to survive intracellularly is still debated, it has been proven that cell-mediated immunity (CMI) plays a pivotal role in protection. In this study we aimed to clarify the interaction ofB. pertussiswith human monocyte-derived dendritic cells (MDDC), evaluating the ability of the bacterium to enter MDDC, to survive intracellularly, to interfere with the maturation process and functional activities, and to influence the host immune responses. The results obtained showed thatB. pertussishad a low capability to be internalized by—and to survive in—MDDC. Upon contact with the bacteria, immature MDDC were induced to undergo phenotypic maturation and acquired antigen-presenting-cell functions. Despite the high levels of interleukin-10 (IL-10) and the barely detectable levels of IL-12 induced byB. pertussis, the bacterium induced maturation of MDDC and T helper 1 (Th1) polarized effector cells. Gene expression analysis of the IL-12 cytokine family clearly demonstrated thatB. pertussisinduced high levels of the p40 and p19 subunits of IL-23 yet failed to induce the expression of the p35 subunit of IL-12. Overall our findings show thatB. pertussis, even if it survives only briefly in MDDC, promotes the synthesis of IL-23, a newly discovered Th1 polarizing cytokine. A Th1-oriented immune response is thus allowed, relevant in the induction of an adequate CMI response, and typical of protection induced by natural infection or vaccination with whole-cell vaccines.

Sequence analysis of the penA gene, encoding penicillin-binding protein 2 (PBP2), in 30 penicillin-intermediate (PenI) Neisseria meningitidis strains showed altered gene sequences due to the translocation of exogenous DNA blocks derived from commensal neisseriae, which are known to have PBP2 proteins with decreased affinity for the antibiotic. In order to obtain a rapid and reproducible method for predicting the PenI phenotype, a real-time PCR assay was set up with primers and probes designed on the basis of the penA gene. The A→G mutation at codon 566, in the transpeptidase domain of the penA gene (which is present in the whole sample of 30 PenI strains and in all the 41 sequences of PenI meningococci isolated worldwide and has been deposited in the sequence databank), was chosen as a marker of penA translocations. Two hybridization probes were designed to distinguish the wild-type penA gene in penicillin-susceptible (PenS) meningococci from the mutated penA gene at codon 566 in PenI strains. Thermal analysis of probe hybridization revealed a melting temperature difference of at least 6°C between PenI and PenS strains. This real-time PCR protocol characterizes the penicillin phenotype of N. meningitidis in a few hours without DNA sequencing and is useful for rapid screening of the penicillin-intermediate genotype among meningococcal isolates.

The relative value of antibodies and/or T-cell immune responses toBordetella pertussisantigens in the immunity induced by acellular pertussis (aP) vaccines is still an open issue, probably due to the incomplete knowledge on the mechanisms of protective immunity to pertussis. The relevance of T-cell immune responses in protection from pertussis has been demonstrated in murine and human models of infection; thus, in this study, the ability of different vaccine preparations of three component (pertussis toxin, filamentous hemagglutinin, and pertactin) aP vaccines to induce T-cell responses was investigated in mice. All vaccine preparations examined passed the immunogenicity control test, based on antibody titer assessment, according to European Pharmacopoeia standards, and protected mice fromB. pertussisintranasal challenge, but not all preparations were able to prime T cells to pertussis toxin, the specificB. pertussisantigen. In particular, one vaccine preparation was unable to induce proliferation and gamma interferon (IFN-γ) production while the other two gave borderline results. The evaluation of T-cell responses to pertussis toxin antigen may provide information on the protective immunity induced by aP vaccines in animal models. Considering the critical role of the axis interleukin-12-IFN-γ for protection from pertussis, our results suggest that testing the induction of a key protective cytokine such as IFN-γ could be an additional tool for the evaluation of the immune response induced by aP vaccines.

The study of antigen specific IgG subclass distribution during disease, or during any other natural or artificial immunisation, can provide useful information on the kind of the immune response and the expected levels of protection. This is particularly true for diseases, such as pertussis in which the mechanisms underlying specific defence are still not completely understood. An investigation was therefore performed to evaluate the IgG subclass response to pertussis toxin (PT) in sera from 89 healthy vaccinated children and 131 vaccinated or unvaccinated children convalescent after a confirmed B. pertussis symptomatic infection. Antibody titres were expressed in arbitrary ELISA units/ml, and statistical analyses were performed. In unvaccinated convalescent children IgG1 and IgG3 were prevalent whereas in children immunised with two different acellular pertussis (aP) vaccines, both healthy and convalescent, IgG1, IgG2 and IgG4 antibodies were mainly produced. Maintenance of the same anti-PT antibody response pattern in healthy acellular pertussis vaccine recipients and in vaccinated children who later acquire the disease is an interesting result indicative of the priming effect induced by these vaccines in the direction of a relatively higher Th2 cell-polarisation of the immune response.

We describe the first reported cases of invasive type e Haemophilus influenzae disease in Italy. All five cases occurred in adults. The isolates were susceptible to ampicillin and eight other antimicrobial agents. Molecular analysis showed two distinct type e strains circulating in Italy, both containing a single copy of the capsulation locus.

The molecular characteristics of 14 B:NT:P1.14 Neisseria meningitidis isolates, collected from 2007 through 2010 in Italy, have been investigated. The B:NT:P1.14 phenotype has only more recently been identified in our country, mainly associated with clonal complex CC162, which is rare in Italy.

The pathogenicity locus (PaLoc) of Clostridium difficile contains toxin A and B genes and three accessory genes, including tcdD and tcdC , which are supposed to code for the positive and negative regulators of toxin expression, respectively. Different studies have described variations in C. difficile toxin A and B genes, but little is known about C. difficile variants for the accessory genes. The PaLoc of several C. difficile clinical isolates was investigated by three different PCR methods with the aim to identify variant strains. Of the toxinogenic C. difficile strains examined, 25% showed variations. No correlation between C. difficile variant strains and key patient groups was found. Interestingly, all of these strains showed a variant tcdC gene. Three different tcdC alleles were identified, and one of these had a nonsense mutation which reduced the TcdC protein from 232 to 61 amino acids. It is possible that different TcdC variants affect toxin production differently, a hypothesis with important implications for the pathogenic potential of variant C. difficile strains.

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.

This study compared a repetitive-element PCR (rep-PCR) method (DiversiLab system) to PCR ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR ribotype 027 isolates tested, different rep types could be distinguished. rep-PCR showed a higher discriminatory power than PCR ribotyping. Nevertheless, this method requires technical skill, and visual interpretation of rep-PCR fingerprint patterns may be difficult.

Comment in: Hidden burden of undiagnosed Clostridium difficile infection. [Lancet Infect Dis. 2014] BACKGROUND: Variations in testing for Clostridium difficile infection can hinder patients' care, increase the risk of transmission, and skew epidemiological data. We aimed to measure the underdiagnosis of C difficile infection across Europe. METHODS: We did a questionnaire-based study at 482 participating hospitals across 20 European countries. Hospitals were questioned about their methods and testing policy for C difficile infection during the periods September, 2011, to August, 2012, and September, 2012, to August, 2013. On one day in winter, 2012-13 (December, 2012, or January, 2013), and summer, 2013 (July or August), every hospital sent all diarrhoeal samples submitted to their microbiology laboratory to a national coordinating laboratory for standardised testing of C difficile infection. Our primary outcome measures were the rates of testing for and cases of C difficile infection per 10 000 patient bed-days. Results of local and national C difficile infection testing were compared with each other. If the result was positive at the national laboratory but negative at the local hospital, the result was classified as undiagnosed C difficile infection. We compared differences in proportions with the Mann-Whitney test, or McNemar's test if data were matched. FINDINGS: During the study period, participating hospitals reported a mean of 65·8 tests (country range 4·6-223·3) for C difficile infection per 10 000 patient-bed days and a mean of 7·0 cases (country range 0·7-28·7) of C difficile infection per 10 000 patient-bed days. Only two-fifths of hospitals reported using optimum methods for testing of C difficile infection (defined by European guidelines), although the number of participating hospitals using optimum methods increased during the study period, from 152 (32%) of 468 in 2011-12 to 205 (48%) of 428 in 2012-13. Across all 482 European hospitals on the two sampling days, 148 (23%) of 641 samples positive for C difficile infection (as determined by the national laboratory) were not diagnosed by participating hospitals because of an absence of clinical suspicion, equating to about 74 missed diagnoses per day. INTERPRETATION: A wide variety of testing strategies for C difficile infection are used across Europe. Absence of clinical suspicion and suboptimum laboratory diagnostic methods mean that an estimated 40 000 inpatients with C difficile infection are potentially undiagnosed every year in 482 European hospitals. Astellas Pharmaceuticals Europe